The nematode Caenorhabditis elegans is one of the most important model organisms for biomedical research, because of its biological tractability and because many of its physiological pathways show strong analogies to corresponding pathways in humans. The goal of this project is to complement the highly developed genomics and proteomics of C. elegans with a comprehensive structural and functional characterization of its metabolome, which, surprisingly, has been explored to only a very limited extent. This effort is motivated by several lines of evidence indicating that small molecules of largely undetermined structure play important roles in C. elegans endocrine and exocrine signaling, specifically in key pathways regulating lifespan, development, and metabolism. Central to the proposed research is the use of new NMR-spectroscopic methodology that permits the analysis of complex small molecule mixtures and greatly accelerates both the structure elucidation process and the functional characterization of the detected compounds. This methodology permits to compare complex metabolite samples derived from different C. elegans mutant strains, and to identify the chemical structures of compounds whose biosynthesis is strongly up- or downregulated as a result of a specific mutation. For this project, a small number of mutant strains were selected whose phenotypes suggest a defect in small-molecule signaling affecting lifespan, development, or fat metabolism. NMR-spectroscopy will be used to detect and identify compounds that correlate with these phenotypes. Subsequently, synthetic samples of the identified compounds will be subjected to chemical genetic screens to determine effects on lifespan, developmental regulation and fat metabolism regulation. Compounds that show activity in wild-type C. elegans will be subjected to additional assays with mutant or RNAi strains representative of key genetic pathways related to ageing and development. Successful conclusion of this project will provide a partial structural and functional annotation of the C. elegans metabolome, substantially increasing our understanding of fundamental pathways in C. elegans biology and corresponding disease-relevant pathways in mammals. The small-molecule knowledge generated will not only enable future efforts aimed at more varied chemical genetic screens exploring additional aspects of the biology and ecology of C. elegans, but also of nematode species relevant in agriculture or medicine. Furthermore, methodology developed for characterizing C. elegans signaling molecules will facilitate similar studies toward structural and functional characterization of small molecule metabolites from other model organisms. PUBLIC HEALTH RELEVANCE: The pervasive physiological changes associated with aging are reflected in age- dependent increases in the incidence of many diseases, including, diabetes, cancer, neurological disorders, heart disease, and osteoporosis. In nematodes, endogenous compounds called ascarosides have been shown to significantly retard the effects of ageing and increase lifespan. The proposed study aims to investigate the biological mechanisms through which these and other endogenous compounds modulate lifespan and development in nematodes, which will contribute to our understanding of the causes of ageing in humans.